Biofilms are a ubiquitous form of growth for bacteria on surfaces in most environments, natural or manmade. Here we present a protocol using the Bioflux microfluidic system to investigate the organized structure and development of these multicellular communities. Microfluidic systems present an opportunity to grow biofilms in a stable, physiologically maintained environment that is readily observable via time-lapse microscopy.
Biofilms are communities of microorganisms that attach to various surfaces and are widely associated with infections. Our investigation is focussed on a current and growing concern: the formation of biofilms in washing machines. Many countries wash clothes at reduced temperatures (30°C to 40°C) rather than at higher temperatures above 60°C that would kill the bacteria. Survival of the bacteria is associated with biofouling, malodour and an increased infection risk due to the distribution of human pathogens such as Pseudomonas aeruginosa, one of the predominant bacteria found in washing machines. Little is known about environmental microniches present in biofilms. Here, we focus on the pH variation throughout P. aeruginosa biofilms knowing that the pH can influence biofilm formation and could be an important aspect for the prevention of biofilms. We use novel pH-sensitive optical nanosensors that penetrate P. aeruginosa biofilms and emit fluorescence in response to pH variation. Using time lapse imaging, pH changes were tracked in real time at a single cell level which will ultimately facilitate monitoring of environmental changes induced as biocides penetrate biofilms. We also look at the isolation and identification of P. aeruginosa from household washing machines. Whole genome analysis was performed to identify different genomic features relevant to antimicrobial resistance (AMR) and biofilm formation. Furthermore, testing of different washing detergent formulations revealed a range of abilities to disrupt biofilm formation or kill P. aeruginosa, which will facilitate the development of more effective washing agents to limit the emergence of AMR within biofilms.
Biofilms are communities of microorganisms that attach to various surfaces and are widely associated with infection for animals and plants. Our investigation is focussed on a current and growing concern: the distribution and formation of biofilms in washing machines. Many countries wash clothes at reduced temperatures around 30 to 40 °C degrees rather than at higher temperatures above 60 °C that would kill the bacteria. Survival of the bacteria is associated with biofouling, malodour and an increased infection risk due to the distribution of human pathogens such as Pseudomonas aeruginosa into the environment. P. aeruginosa is one of the predominant bacteria found in washing machines and is highly resistant to many antibiotics. Little is known about environmental microniches present in biofilms. In this work, we focus on the pH variation throughout P. aeruginosa biofilms knowing that the pH can influence biofilm formation and could be an important aspect for the prevention of biofilm formation. Here, we use novel pH-sensitive optical nanosensors that penetrate P. aeruginosa biofilms and emit fluorescence in response to variation in pH. Confocal laser scanning microscopy revealed that the nanosensors can penetrate biofilms within minutes and interact with the biofilm structure. Different washing detergents were tested resulting in altered biofilm formation and killing abilities. Using time lapse imaging, pH changes were tracked in real time at a microcolony and single cell level which will ultimately facilitate monitoring of environmental changes induced as biocides penetrate biofilms, underpinning the development of more effective antimicrobials to limit the emergence of AMR.
Streptococcus mutans is a pre-dominant bacterial species found in oral biofilms and participates in the production of dental caries via the generation of organic acids. The production of these acids results from the fermentation of carbohydrates present in a sugar-laden diet. As the acidity of an oral biofilm decreases, the demineralisation of the enamel of a tooth increases; leading to the formation of dental caries. To detect and measure the pH change occurring following a sugar challenge, ratiometric, fluorescent, pH-sensitive nanosensors were incorporated into oral biofilms. Confocal laser scanning microscopy revealed that the addition of glucose (1 % w/v) to an S. mutans biofilm resulted in a gradual reduction in the fluorescence intensity ratio during a 30 min period. This reduction in the fluorescence intensity ratio indicated a reduction in pH of the biofilm over time as the glucose was being fermented, resulting in the production and secretion of acids into the extracellular matrix of the biofilm. Additionally, a reduction in pH was detected – using widefield microscopy – in starved, planktonic S. mutans when treated with glucose. Over the course of 30 min, the pH of the medium was reduced from pH 5.3 to pH 3.3 as the glucose was fermented by the bacteria. These findings will help us map pH changes in oral biofilms as we examine potential methods of preventing the acidification of oral biofilms and the eventual demineralisation of the enamel; leading to the reduction in dental caries and an improvement in the standard of living of those effected.
Understanding the dynamic environmental microniches of biofilms will permit us to detect, manage and exploit these communities. The components and architecture of biofilms have been interrogated in depth, however, little is known about the environmental microniches present. This is primarily because of the absence of tools with the required measurement sensitivity and resolution to detect these changes. We describe the application of ratiometric fluorescent pH-sensitive nanosensors, as a novel tool, to observe physiological pH changes in biofilms in real-time. Nanosensors comprised two pH-sensitive fluorophores covalently encapsulated with a reference pH-insensitive fluorophore in an inert polyacrylamide nanoparticle matrix. The nanosensors were used to analyse the real-time three-dimensional pH variation for two model biofilm formers: (i) opportunistic pathogen Pseudomonas aeruginosa, and (ii) oral pathogen Streptococcus mutans. The detection of sugar metabolism in real time by nanosensors provides a potential application to identify novel therapeutic solutions to improve oral health.
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